This invention relates to a method and apparatus for performing small scale reactions. In particular, the instant disclosure pertains to small scale cycling reactions, isothermal reactions, and devices for assembly of sub-microliter reaction mixtures.
The Human Genome Program is a scientific endeavor that is a national priority of the United States. The original goal of the federally funded U.S. effort had been to complete the sequence at ten-fold coverage by the year 2005. A draft five-fold deep version of the human genome will now be produced by the year 2001. To accomplish this goal, the effort has accelerated to improve sequencing throughput rates and reduce DNA sequencing costs.
In the late 1970s, Sanger et al. developed an enzymatic chain termination method for DNA sequence analysis that produces a nested set of DNA fragments with a common starting point and random terminations at every nucleotide throughout the sequence. Lloyd Smith, Lee Hood, and others modified the Sanger method to use four fluorescent labels in sequencing reactions enabling single lane separations. This resulted in the creation of the first automated DNA sequencers that used polyacrylamide slab gels. More recently, fluorescent energy-transfer dyes have been used to make dye sets that enhance signals by 2- to 10-fold and simplify the optical configuration.
Automated fluorescent capillary array electrophoresis (CAE) DNA sequencers appear to be the consensus technology to replace slab gels. Capillary gel electrophoresis speeds up the separation of sequencing products and has the potential to dramatically decrease sample volume requirements. The 96-channel CAE instrument, MegaBACE(trademark), which is commercially available from Molecular Dynamics (Sunnyvale, Calif.), uses a laser-induced fluorescence (LIF) confocal fluorescence scanner to detect up to an average of about 625 bases per capillary (Phred 20 window) in 90 minute runs with cycle times of two hours. Confocal spatial filtering results in a higher signal-to-noise ratio because superfluous reflections and fluorescence from surrounding materials are eliminated before signal detection at the photomultiplier tube (PMT). Accordingly, sensitivity at the level of subattomoles per sequencing band is attainable. Confocal imaging is also particularly important in capillary electrophoresis in microchip analysis systems where the background fluorescence of a glass or plastic microchip may be much higher than that of fused silica capillaries. Capillary array electrophoresis systems will solve many of the initial throughput needs of the genomic community for DNA analysis. However, low volume sample preparation still presents a significant opportunity to increase throughput and reduce cost.
While fluorescent DNA sequencers are improving the throughput of DNA sequence acquisition, they have also moved the throughput bottleneck from sequence acquisition back towards sample preparation. In response, rapid methods for preparing sequencing templates and for transposon-facilitated DNA sequencing have been developed as have magnetic bead capture methods that eliminate centrifugation. Thermophilic Archae DNA polymerases have been screened and genetically engineered to improve fidelity, ensure stability at high temperatures, extend lengths, and alter affinities for dideoxynucleotides and fluorescent analogs. These improvements have resulted in lower reagent costs, simpler sample preparation, higher data accuracy, and increased readlengths.
The sequencing community has also developed higher throughput methods for preparing DNA templates, polymerase chain reaction (PCR) reactions, and DNA sequencing reactions. Sample preparation has been increasingly multiplexed and automated using 96- and 384-well microtiter plates, multi-channel pipettors, and laboratory robotic workstations. In general, these workstations mimic the manipulations that a technician would perform and have minimum working volumes of about a microliter, although stand-alone multi-channel pipettors are being used to manipulate smaller volumes.
A typical full-scale sample preparation method for DNA shotgun sequencing on capillary systems begins by lysing phage plaques or bacterial colonies to isolate subcloned DNA. Because capillary electrophoresis is more sensitive to impurities in sequencing reactions than slab gels, the subcloned DNA insert is frequently PCR-amplified to exponentially increase its concentration in the sample. Next, exonuclease I (ExoI) and arctic shrimp alkaline phosphatase (SAP) are added to perform an enzymatic cleanup reaction to remove primer and excess dNTPs that interfere with cycle sequencing. ExoI is used to degrade the single-stranded primers to dNMPs without digesting double-stranded products. SAP converts dNTPs to dNMPs and reduces the DNTP concentration from 200 xcexcM, as used for the PCR reaction, to less than 0.1 xcexcM for use with fluorescent sequencing. The reaction is performed at 37xc2x0 C. and then heated to 65xc2x0 C. to irreversibly denature the ExoI and SAP.
Because the PCR amplification produces excess template DNA for cycle sequencing, the ExoI/SAP treated PCR sample can be diluted five-fold before cycle sequencing. This reduces the concentration of contaminants into a range that causes less interference with CAE analysis. Cycle sequencing reagents are added, typically with fluorescently labeled dye primers or terminators and the reaction is thermal cycled to drive linear amplification of labeled fragments. Finally, after cycling, the samples are ethanol precipitated, resuspended in formamide, another denaturant, or water, and the sample is electrokinetically injected into the CAE system.
This workflow has resulted in a dramatic improvement in the performance of the MegaBACE system and similar workflows currently appear to be the methods of choice for other CAE systems as well. Using actual samples from single plaques and colonies of human genomic random subclones or Expressed Sequence Tags (ESTs), this workflow with linear polyacrylamide as a separation matrix has improved the success rate of samples over 200 base pairs from about 60% to 85-90%, and has improved the average readlength from about 400 to greater than 600 bases. Furthermore, this method has proven to be quite robust.
While the above sample preparation methods have greatly increased throughput, the cost of reagents remains a major component of the cost of sequencing. CAE requires only subattomoles of sample, but presently samples are prepared in the picomole range. Reducing the reaction volume will therefore reduce the cost of DNA sequencing and still provide enough material for analysis. However, substantial reductions in reaction volume can only be achieved if satisfactory methods can be developed for manipulating and reacting samples and reagents. Ideally, such a method would be automated and configured in order that multiple samples could be produced at one time. Moreover, it would be desirable to integrate such a method as a module capable of interfacing with additional components, such as CAE and a detector for separation and analysis.
Several devices have been designed to aid in the automation of sample preparation. For example, U.S. Pat. No. 5,720,923 describes a system in which small scale cycling reactions take place in tubes with diameters as small as 1 mm. The tubes are subsequently exposed to thermal cycles produced by thermal blocks to effect the desired reaction. Multiple samples may be processed in a single tube by drawing in small amounts of sample, each of which are separated in the tube by a liquid which will not combine with the sample. Fluid moves through the tubes by means of a pump. These features are incorporated into a system which automatically cleans the tubes, moves sample trays having sample containing wells, and brings the tubes into contact with the wells in the sample trays.
U.S. Pat. No. 5,785,926 discloses a system for transporting small volumes of sample. In this system, at least one capillary tube is used to transport small amounts of sample. A precision linear actuator connected to a computer controlled motor acts as a pneumatic piston to aliquot and dispense liquid using the tube. The sample amount is monitored by an optical sensor that detects the presence of liquid within the capillary segment. The system includes a fluid station containing liquids to be deposited and a positioning device for positioning the transport capillary.
U.S. Pat. No. 5,897,842 discloses a system for automated sample preparation using thermal cycling. In this system a reaction mixture is pumped into a capillary tube. One end of the tube is sealed using pressure from an associated pump while the other end is sealed by pressing the tube against a barrier. The pump also serves to move fluid within the tube. Once the ends are sealed, the tube is exposed to thermal cycles. In this system a robotic transfer device moves the tubes between the sample preparation station where the pump loads the components of the reaction mixture into the tubes and the thermal cycling station.
There is an additional need for an automated system that is able to perform small-scale thermal cycling reactions in a highly parallel manner. The system should allow for rapid preparation of cycling reactions with minimal consumption of reagents. The combination of reducing the amount of reagents required for a reaction and reducing the time required for a reaction will greatly reduce the overall cost of preparation of cycling reactions.
Capillary array electrophoresis systems and capillary electrophoresis microchip analytical systems can detect subattomoles of reaction products. It is one object of the invention to disclose a method and system for cycling reactions that operate on a submicroliter scale that takes advantage of the high sensitivity of these analytical systems. This reduction of reaction volume will lower the reagent requirements and cost of each reaction. It is a further object to provide an automated system that is able to reduce the time required for cycling reaction preparation. It is an additional object of the invention to provide a system that may be integrated with analytical instruments including capillary array electrophoresis systems and electrophoresis chips.
It is a further object of the invention to provide an automated system for preparing reactions and filling reaction containers using capillary action. This allows metering a quantity of liquid into a capillary tube length of fixed volume without using external force to pump liquids. It is a further object to disclose a reagent-metering device that also may act as the reaction container. It is also an object of the invention to provide a system that allows the nanoscale reaction containers to be cleaned and reused, saving material costs.
It is a further object of the invention to provide a system with highly parallel processing, allowing greater throughput. Preferably, the system would match the density of micro-well plates. It is also an object of the invention to have an automated system in which a number of different cycling reactions could be performed in parallel using a single temperature regulation source, allowing more efficient use of the thermal cycling apparatus. It is a further object to perform isothermal reactions in a highly parallel manner in submicroliter volumes. It is also an object of the invention to provide an automated reaction preparation system that is able to utilize available automation tools by being compatible with standard plate size formats.
The above objects have been achieved through a system and method for preparing cycling reaction mixtures. The system uses a capillary cassette comprised of a number of capillary tube segments arranged in parallel alignment. The tube segments extend through a substrate and are generally positioned with uniform spacing. The capillary cassette may be used both to meter reagents and as a reaction chamber in which the reaction is conducted.
A reaction mixture containing a nucleic acid sample and reaction reagents for performing a thermal cycling reaction (such as the polymerase chain reaction, ligase chain reaction, or chain termination sequencing reaction) is introduced into the capillaries of a capillary cassette. In one embodiment each capillary contains a unique nucleic acid sample but the same reaction reagents.
The reaction mixture may be generated in various manners. In one sample preparation method, sample DNA adheres to the interior of the capillary tubes of the capillary cassette or onto a substrate. The liquid in which the DNA was suspended may be eliminated from the capillary or substrate while the nucleic acid is retained, bound to the capillary or substrate. The reaction reagents may then be introduced into the capillary or substrate, combining the sample and reaction reagents to form an assay mixture. In another sample preparation method, the capillaries in a capillary cassette or the wells in a multiwell plate are coated with dehydrated reaction reagents. The nucleic acid sample is introduced into the capillaries of the capillary cassette or the wells of a multiwell plate and the nucleic acid sample rehydrates the reaction reagents to form a reaction mixture. If the multiwell plate is used, the reaction mixture is subsequently transferred into the capillaries of a capillary cassette. In another sample preparation method, both the reaction reagents and the nucleic acid samples are metered by the capillaries of a capillary cassette. The capillaries are dipped into the wells of a sample plate and a fixed amount of fluid (defined by the interior volume of the capillary) is drawn into the capillary. The volume of liquid metered by the capillary tubes is dispensed by positive displacement, centrifugal force, or other displacement method into the wells of a microplate. A capillary cassette is used to meter both the reaction reagents in a similar manner and dispense the metered liquids onto a location on a substrate combining the sample and reaction reagents to form a reaction mixture. In any of these reaction mixture preparation methods, reaction reagents, nucleic acid samples and assembled reaction mixtures are introduced into the capillary tubes of a capillary cassette or drawn into the capillary cassette by capillary action. Liquids may also be introduced into the capillaries by active filling, such as by pressure or vacuum. For example, one end of the capillaries may be sealed with a liquid impermeable (hydrophobic), gas permeable membrane. By applying a vacuum force to one side of the membrane, the capillary will fill with liquid to the level of the membrane, where hydrophobic forces will prevent further filling of the capillary.
The capillary cassette filled with the reaction mixture is next sealed by pressing the two ends of the capillary tube segments against deformable membranes. The capillary cassette with ends sealed against deformable membranes is contained within an interior chamber of a temperature cycling device. The temperature cycling device exposes the contents of the capillaries to thermal cycles, causing the thermal cycling reaction to occur. In one embodiment the thermal cycling apparatus is an air thermal cycling device. This device receives the capillary cassette into an interior chamber where the ends of the capillaries in the cassette are sealed. The temperature changes occur using rapidly flowing air. The temperature of the cycling air may be rapidly lowered by venting air to outside the interior cycling chamber. A thermocouple sensor in the air path of the capillary cassette allows for precise monitoring of the temperature of the reaction mixture. Given the rapid transfer of heat through the capillary and precise temperature sensing allowed by the thermocouple, rapid reaction times are possible. The complete thermal cycling times needed for 30 cycles of denaturing heating followed by a period of lower temperature for extension of a 600-700 base DNA strand can be performed in 30 minutes or less and could theoretically be effected in as little as 8 minutes. Following a programmed number of thermal cycles, the capillary cassette is removed from the temperature cycling chamber.
The reaction mixture is next dispensed from the capillary cassette and transferred into a substrate. In one embodiment the substrate into which the completed reaction mixture is dispensed is an analytical chip, such as an electrophoresis chip. Following transfer from the capillary cassette, the reaction mixture may be separated and analyzed. Alternatively, the sample may be dispensed into a microplate or other substrate. The substrate may then be placed, manually or by an automated system, in a location where it may be analyzed by capillary array electrophoresis. In addition to electrophoresis, the instant reaction preparation system may also be adapted for use in preparing nucleic acid, protein or other biomolecules for microarray analysis, mass spectrometry analysis, or other analysis methods. The capillary cassette may also be used for conducting ELISA or other assays requiring binding to a substrate.
The use of the present system allows a simplified transition between nanoscale and larger scale preparation steps. For example, the PCR step may be performed in the nanoscale in the capillary cassette of the present invention. The resulting products can be dispensed into a microplate well for enzymatic clean up on a larger scale such as microliter volumes. Following clean up, the amplified nucleic acid can again be metered into a nanoscale capillary cassette for subsequent reaction mixture preparation (e.g. cycle sequencing). This achieves a simple transition method from the nanoscales into larger scales.
Depositing the reaction mixtures from the capillary cassette into the wells of a 96 well plate allows subsequent analysis by the capillary array electrophoresis systems. Post reaction processing is also possible. This could include depositing the reaction mixture into ethanol to precipitate the DNA fragments produced in the reaction or dispensing the reaction mixture into formamide to denature double stranded DNA reaction products.
Following each use, the capillary cassette may be placed into a capillary cassette washer and washed. Following washing, the capillary cassette may be reused.
The system can be designed with magazines for holding the sample plates, the multi-well mixing plates, and the plates containing the finished reactions. This would allow the system to operate continuously to prepare reaction mixtures. In addition, an integrated system with a central electronic control would allow for a system that may simultaneously assemble reaction mixtures, perform thermal cycling or other reactions, and wash capillary cassettes.
The system is useful for the preparation of sequencing reactions, but may also be used in highly parallel preparation of cell lysates, plasmid extraction, polymerase chain reactions, ligase chain reactions, rolling circle amplification reactions, screening compound libraries for drug discovery or compound activity, protein digestion/sequencing, ELISA, radioimmunoassays and other chemical or biochemical reactions or assays.